METHOD FOR SAVING AN IMAGE ACQUIRED USING SINGLE MOLECULE LOCALIZATION MICROSCOPY

DETAILED ACTION Notice of Pre-AIA or AIA Status The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . Response to Arguments The reply filed on 8/28/2026 has been entered. Applicant’s arguments with respect to claims 1-8 have been considered but are moot in view of new ground(s) of rejection caused by the amendments. Claims 1-8 are pending in this application and have been considered below. Priority Receipt is acknowledged that application is a National Stage application of PCT/EP2023/050920. Priority to EP22152746.8 with a priority date of 1/21/2022 is /acknowledged under 35 USC 119(e) and 37 CFR 1.78. Information Disclosure Statement The IDSs dated 7/19/2024 and 8/01/2024 that have been previously considered remain placed in the application file. 1st Claim Rejections - 35 USC § 103 In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action: A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made. The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows: 1. Determining the scope and contents of the prior art. 2. Ascertaining the differences between the prior art and the claims at issue. 3. Resolving the level of ordinary skill in the pertinent art. 4. Considering objective evidence present in the application indicating obviousness or nonobviousness. This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention. Claims 1, 2, 4, 5, 6, and 8 are rejected under 35 U.S.C. 103 as obvious over US Patent Publication 2011 0175995 A1, (Doerrer) in view of EP Patent Publication 2020 3660490 A1, (Garcia-Parajo et al.). Claim 1 [AltContent: textbox (Figure 2 shows the detected spots from the scanned sample.)] PNG media_image1.png 382 520 media_image1.png Greyscale Regarding claim 1, Doerrer teaches a method for saving an image, the method comprising: a) acquiring an image of a sample; ("such a method comprises scanning the slide so as to determine objects of interest in the sample," par. 14) b) detecting a plurality of spots in the acquired image; c) determining one or more local regions of interest of the acquired image comprising the detected plurality of spots; ("determine objects of interest in the sample," par. 14) d) determining metadata defining each of the one or more local regions of interest; ("determine objects of interest in the sample, associating corresponding coordinates with each object of interest," par. 14) e) creating one or more image files, wherein each of the one or more image files comprises image data of each of the one or more local regions of interest and the determined metadata defining each of the one or more local regions of interest, wherein the image data is a portion of the acquired image; and f) saving the one or more image files ("storing an image and corresponding coordinates of each object of interest," par. 14). Doerrer does not explicitly teach all of an image acquired using single molecule localization microscopy, a sample comprising a plurality of single fluorescent molecules using single molecule localization microscopy, and each spot representing one single fluorescent molecule of the plurality of single fluorescent molecules. However, Garcia-Parajo et al. teach an image acquired using single molecule localization microscopy, a sample comprising a plurality of single fluorescent molecules using single molecule localization microscopy, and each spot representing one single fluorescent molecule of the plurality of single fluorescent molecules ("individual fluorescent molecules can be imaged and their spatial coordinates can be localized and determined with very high precision," par. 2). Therefore, taking the teachings of Doerrer and Garcia-Parajo et al. as a whole, it would have been obvious to a person having ordinary skill in the art before the time of the effective filing date of the claimed invention of the instant application to modify spot detection as taught by Doerrer to use the single molecule localization microscopy techniques and image reconstruction methods as taught by Garcia-Parajo et al. The suggestion/motivation for doing so would have been that, “The method of the current application applied to single-molecule localization microscopy techniques can enable to obtain a gain in throughput and imaging speed. The known state of the art DNA-PAINT single-molecule localization microscopy technique combined with the frequency-modulated illumination, which can also be called frequency-modulated-DNA-Paint or fm-DNA-PAINT, can enable to obtain an increase of gain in imaging throughput and improved imaging speed” as noted by the Garcia-Parajo et al. disclosure in paragraph [0020], which also motivates combination because the combination would predictably have a greater efficiency as there is a reasonable expectation that by extracting and storing only the precise spatial coordinates and intensity data, the volume of data required for image reconstruction is minimized, thereby reducing storage overhead and optimizing computational efficiency during final super-resolution rendering; and/or because doing so merely combines prior art elements according to known methods to yield predictable results. Claim 2 Regarding claim 2, Doerrer and Garcia-Parajo et al. teach the method according to claim 1 as noted above. Doerrer teaches the method according to claim 1, wherein the method is repeated multiple times, and each time the method is repeated, the method comprises repeating steps a) to e) and: g) creating further one or more image files, wherein each of the further one or more image files comprises further image data of each of further one or more local regions of interest and further metadata defining each of the further one or more local regions of interest ("a method comprises scanning the slide so as to determine objects of interest in the sample, associating corresponding coordinates with each object of interest, and storing an image and corresponding coordinates of each object of interest," par. 14 and fig. 2 wherein the process is repeated until all objects of interest have been processed). Doerrer does not explicitly teach all of h) creating a final file comprising the image data of each of the one or more image files and the further image data of the further one or more image files. However, Garcia-Parajo et al. teach h) creating a final file comprising the image data of each of the one or more image files and the further image data of the further one or more image files ("the processing means can be configured to reconstruct an image of the fluorescence emitting elements in the target based on the determined individual fluorescence elements localizations. A super-resolution image with high resolution, or high image resolution, can be reconstructed with the current invention," par. 64). Doerrer and Garcia-Parajo et al. are combined as per claim 1. Claim 4 Regarding claim 4, Doerrer and Garcia-Parajo et al. teach the method according to claim 1 as noted above. Doerrer teaches wherein each of the one or more local regions have a shape which is a polygonal shape and/or an elliptical shape, (See fig. 2, object of interest (330) has an elliptical shape) and the metadata define the position of the one or more local regions in the image ("coordinates in any other suitable coordinate system, of the stored objects of interest with respect to a reference point on the slide are determined and stored in association with the respective image," par. 26 wherein the coordinates). Doerrer does not explicitly teach all of the metadata define the shape of the one or more local regions in the image. However, Garcia-Parajo et al. teach the metadata define the shape of the one or more local regions in the image ("individual fluorescent molecules can be imaged and their spatial coordinates can be localized and determined with very high precision," par. 2). Doerrer and Garcia-Parajo et al. are combined as per claim 1. Claim 5 Regarding claim 5, Doerrer teaches an apparatus for saving and image, the apparatus comprising: an image acquisition unit configured to acquire an acquired image of a sample ("such a method comprises scanning the slide so as to determine objects of interest in the sample," par. 14); a detection unit configured to detect a plurality of spots in the image (See fig. 2); a determination unit configured to determine one or more local regions of interest of the acquired image comprising the detected plurality of spots ("determine objects of interest in the sample," par. 14); a calculation unit configured to determine metadata defining each of the one or more local regions of interest ("determine objects of interest in the sample, associating corresponding coordinates with each object of interest," par. 14); and a file unit configured to create one or more image files and save the one or more image files, wherein each of the one or more image files comprises image data of each of the one or more local regions of interest and the determined metadata defining each of the one or more local regions of interest, wherein the image data is a portion of the acquired image ("storing an image and corresponding coordinates of each object of interest," par. 14). Garcia-Parajo et al. does not explicitly teach all of an image acquired using single molecule localization microscopy, a sample comprising a plurality of single fluorescent molecules, and each spot representing one single fluorescent molecule of the plurality of single fluorescent molecules. However, Garcia-Parajo et al. teach an image acquired using single molecule localization microscopy, a sample comprising a plurality single fluorescent molecules, and each spot representing one single fluorescent molecule of the plurality of single fluorescent molecules ("individual fluorescent molecules can be imaged and their spatial coordinates can be localized and determined with very high precision," par. 2). Doerrer and Garcia-Parajo et al. are combined as per claim 1. Claim 6 Regarding claim 6, Doerrer and Garcia-Parajo et al. teach the apparatus according to claim 5 as noted above. Doerrer teaches wherein the image acquisition unit is configured to acquire further images of the sample, and for each of the acquired further images, the file unit is configured to: create further one or more image files, wherein each of the further one or more image files comprises further image data of each of further one or more local regions of interest and further metadata defining each of the further one or more local regions of interest ("a method comprises scanning the slide so as to determine objects of interest in the sample, associating corresponding coordinates with each object of interest, and storing an image and corresponding coordinates of each object of interest," par. 14 and fig. 2 wherein the process is repeated until all objects of interest have been processed). Doerrer does not explicitly teach all of create a final file comprising the image data of each of the one or more image files and the further image data of the further one or more image files. However, Garcia-Parajo et al. teach create a final file comprising the image data of each of the one or more image files and the further image data of the further one or more image files ("the processing means can be configured to reconstruct an image of the fluorescence emitting elements in the target based on the determined individual fluorescence elements localizations. A super-resolution image with high resolution, or high image resolution, can be reconstructed with the current invention," par. 64). Doerrer and Garcia-Parajo et al. are combined as per claim 1. Claim 8 Regarding claim 8, Doerrer and Garcia-Parajo et al. teach the apparatus according to claim 5 as noted above. Doerrer teaches wherein each of the one or more local regions have a shape which is a polygonal shape and/or an elliptical shape, (See fig. 2, object of interest (330) has an elliptical shape) and the metadata define a position of the one or more local regions in the image ("coordinates in any other suitable coordinate system, of the stored objects of interest with respect to a reference point on the slide are determined and stored in association with the respective image," par. 26 wherein the coordinates). Doerrer does not explicitly teach all of the metadata define the shape of the one or more local regions in the image. However, Garcia-Parajo et al. teach the metadata define the shape of the one or more local regions in the image ("individual fluorescent molecules can be imaged and their spatial coordinates can be localized and determined with very high precision," par. 2). Doerrer and Garcia-Parajo et al. are combined as per claim 1. 2nd Claim Rejections - 35 USC § 103 Claims 3 and 7 are rejected under 35 U.S.C. 103 as obvious over US Patent Publication 2011 0175995 A1, (Doerrer) and EP Patent Publication 2020 3660490 A1, (Garcia-Parajo et al.) in view of US Patent Publication 2002 0081014 A1, (Ravkin). Claim 3 Regarding claim 3, Doerrer and Garcia-Parajo et al. teach the method according to claim 2 as noted above. Doerrer teaches wherein the method is performed at least three times wherein a first image, a second image and a third image are acquired at step a) and the steps b) to h) are performed for each of the first image, the second image and the third image, and the method comprises: ("a method comprises scanning the slide so as to determine objects of interest in the sample, associating corresponding coordinates with each object of interest, and storing an image and corresponding coordinates of each object of interest," par. 14 and fig. 2 wherein the process is repeated until all at least three objects of interest have been processed). Doerrer does not explicitly teach all of i) determining a background of the second image based on the further one or more regions of interest determined for each of the first image, the second image and the third image; j) subtracting the background of the second image; and k) creating a background-free image file comprising image data of the second image without the background. However, Ravkin teach i) determining a background of the second image based on the further one or more regions of interest determined for each of the first image, the second image and the third image ("The sample is then illuminated and imaged in a manner that the different features of interest can be distinguished from each other and from the background," par. 3); j) subtracting the background of the second image; and k) creating a background-free image file comprising image data of the second image without the background ("For the fluorescence image, subtract the background intensity value from all pixels, thereby bringing the fluorescence peak down to mid-gray, bringing the background down to a black or near-black level, and clipping the cytoplasm regions to black," par. 69). Therefore, taking the teachings of Doerrer, Garcia-Parajo et al., and Ravkin as a whole, it would have been obvious to a person having ordinary skill in the art before the time of the effective filing date of the claimed invention of the instant application to modify spot detection as taught by Doerrer and the single molecule localization microscopy techniques and image reconstruction methods as taught by Garcia-Parajo et al. to use background subtraction methods as taught by Ravkin. The suggestion/motivation for doing so would have been that, “For the fluorescence image, subtract the background intensity value from all pixels, thereby bringing the fluorescence peak down to mid-gray, bringing the background down to a black or near-black level, and clipping the cytoplasm regions to black” as noted by the Ravkin disclosure in paragraph [0069], which also motivates combination because the combination would predictably have a higher accuracy as there is a reasonable expectation that subtracting the background would isolate and enhance the target fluorescent intensities, making them more visible and distinct from surrounding noise; and/or because doing so merely combines prior art elements according to known methods to yield predictable results. Claim 7 Regarding claim 7, Doerrer and Garcia-Parajo et al. teach the apparatus according to claim 5 as noted above. Doerrer teaches wherein the image acquisition unit is configured to acquire a first image, a second image and a third image ("a method comprises scanning the slide so as to determine objects of interest in the sample, associating corresponding coordinates with each object of interest, and storing an image and corresponding coordinates of each object of interest," par. 14 and fig. 2 wherein the process is repeated until all at least three objects of interest have been processed). Doerrer does not explicitly teach all of the determination unit is configured to determine a background of the second image based on the further one or more regions of interest determined for each of the first image, the second image and the third image; the determination unit is configured to subtract the background of the second image; and the file unit is configured to create a background-free image file comprising image data the second image without the background. However, Ravkin teach the determination unit is configured to determine a background of the second image based on the further one or more regions of interest determined for each of the first image, the second image and the third image ("The sample is then illuminated and imaged in a manner that the different features of interest can be distinguished from each other and from the background," par. 3); the determination unit is configured to subtract the background of the second image; and the file unit is configured to create a background-free image file comprising image data the second image without the background ("For the fluorescence image, subtract the background intensity value from all pixels, thereby bringing the fluorescence peak down to mid-gray, bringing the background down to a black or near-black level, and clipping the cytoplasm regions to black," par. 69). Doerrer, Garcia-Parajo et al., and Ravkin are combined as per claim 3. Reference Cited The prior art made of record and not relied upon is considered pertinent to applicant’s disclosure. US Patent Publication 2016 0124208 A1 to Best et al. discloses obtaining super-resolution images of a sample labeled with at least one fluorescent label by combining localization microscopy and structured illumination microscopy is provided. Conclusion Any inquiry concerning this communication or earlier communications from the examiner should be directed to KARSTEN F LANTZ whose telephone number is (571) 272-4564. The examiner can normally be reached Monday-Friday 8:00-4:00. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ms. Jennifer Mehmood can be reached on 571-272-2976. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /Karsten F. Lantz/Examiner, Art Unit 2664 Date: 9/30/2026 /PING Y HSIEH/Primary Examiner, Art Unit 2664